dokumen perencanaan skpd BAB (10)

European Journal of Pharmacology 727 (2014) 158–166

Contents lists available at ScienceDirect

European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar

Modulation of peripheral Na þ channels and neuronal firing
by n-butyl-p-aminobenzoate
Olivier Thériault a, Hugo Poulin a, Adrian Sculptoreanu b,1, William C. de Groat b,
Micheal E. O’Leary c, Mohamed Chahine a,n
a

Le Centre de recherche de l’Institut universitaire en santé mentale de Québec, Université Laval, Quebec City, QC, Canada
Department of Pharmacology & Chemical Biology, University of Pittsburgh Medical School, Pittsburgh, PA, USA
c
Department of Biomedical Sciences, Cooper Medical School of Rowan University, Camden, NJ, USA
b

art ic l e i nf o

a b s t r a c t

Article history:
Received 10 October 2013
Received in revised form
15 January 2014
Accepted 22 January 2014
Available online 30 January 2014

n-butyl-p-aminobenzoate (BAB), a local anesthetic, is administered epidurally in cancer patients to treat
pain that is poorly controlled by other drugs that have a number of adverse effects. The purpose of
the study was to unravel the mechanisms underlying the apparent selective pain suppressant effect
of BAB. We used the whole-cell patch-clamp technique to record Na þ currents and action potentials
(APs) in dissociated, nociceptive dorsal root ganglion (DRG) cells from rats, two types of peripheral
sensory neuron Na þ channels (Nav1.7 and Nav1.8), and the motor neuron-specific Na þ channel (Nav1.6)
expressed in HEK293 cells. BAB (1–100 μM) inhibited, in a concentration-dependent manner, the
depolarization evoked repetitive firing in DRG cells, the three types of Na þ current expressed in
HEK293 cells, and the TTXr Na þ current of the DRG neurons. BAB induced a use-dependent block that
caused a shift of the inactivation curve in the hyperpolarizing direction. BAB enhanced the onset of slow
inactivation of Nav1.7 and Nav1.8 currents but not of Nav1.6 currents. At clinically relevant concentrations

(1–100 μM), BAB is thus a more potent inhibitor of peripheral TTX-sensitive TTXs, Nav1.7 and TTXresistant NaV1.8 Na þ channels than of motor neuron axonal Nav1.6 Na þ channels. BAB had similar effects
on the TTXr Na þ channels of rat DRG neurons and Nav1.8 channels expressed in HEK293 cells. The
observed selectivity of BAB in treating cancer pain may be due to an enhanced and selective
responsiveness of Na þ channels in nociceptive neurons to this local anesthetic.
& 2014 Elsevier B.V. All rights reserved.

Keywords:
Dorsal root ganglia
Sodium channels
Nociception
Butamben
Nav
Chemical compounds studied in this article:
Butamben (PubChem CID: 2482)

1. Introduction
The pharmacological management of chronic pain is often
limited by the occurrence of side effects and the development of
dependence and/or tolerance (Dworkin et al., 2007). However, the
epidural administration of n-butyl-p-aminobenzoate (BAB) has

been shown to produce long-lasting (Z 4 weeks) analgesia to
chronic pain patients with no significant adverse effects or loss of
motor function (McCarthy et al., 2002;Shulman et al., 1998, 2000).
These observations raised the possibility that BAB inhibits subtypes of voltage gated sodium channels (VGSCs) in peripheral
neurons and the spinal cord with different affinities. This idea was

Abbreviations: AP, action potential; BAB, n-butyl-p-aminobenzoate; VGSCs, voltage gated Na þ channels; OS, Overshoot; TTXr, TTX-resistant; TTXs, TTX-sensitive
n
Correspondence to: Le Centre de recherche de l’Institut universitaire en santé
mentale de Québec, 2601 chemin de la Canardière, Quebec City, QC, Canada G1J
2G3. Tel.: þ1 418 663 5747x4723; fax: þ 1 418 663 8756.
E-mail address: mohamed.chahine@phc.ulaval.ca (M. Chahine).
1
Present address: Neurobiology of Learning Unit, Universita' Vita-Salute San
Raffaele, Milan, Italy
0014-2999/$ - see front matter & 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2014.01.036

supported by the fact that BAB has different inhibitory effects
on tetrodotoxin-resistant(TTXr) and tetrodotoxin-sensitive (TTXs)

Na þ channels in isolated rat DRG neurons (Van den Berg
et al., 1995, 1996). The interpretation of the findings from these
early studies is complicated by the many Na þ channel subtypes
expressed in afferent neurons. The interpretation of the results is
made even more challenging by the difficulty in determining the
exact identity of Na þ channel subtypes using pharmacological and
electrophysiological methods.
Various Na þ channel subtypes are upregulated and their
subcellular distribution is changed following nerve injury or
inflammation, which results in chronic pain. Hyperexcitability in
primary afferents induced by noxious stimuli occurs in several
models of nerve injury, inflammation, and nociceptive sensitization (Moore et al., 2002;Song et al., 2003) due to changes in the
kinetics, voltage-dependence, and expression of VGSCs (Black
et al., 2004;Moore et al., 2002;Gold and Flake, 2005;Gold et al.,
1998, 2003;Thakor et al., 2009). In addition, the inhibition of
K þ channels has also been implicated in the induction of both
acute (Sculptoreanu and De Groat, 2007) and chronic nociceptive
sensitization (Sculptoreanu et al., 2005).

O. Thériault et al. / European Journal of Pharmacology 727 (2014) 158–166

In the present study, we used the patch-clamp method to
investigate the actions of BAB on the properties of sensory neuron
Na þ channel subtypes, TTXs Nav1.7, TTXr Nav1.8, and the motor
neuron axonal subtype Nav1.6 stably transfected in HEK293 cells.
We compared the effects on these channels to the effects on TTXr
Na þ channels in a select group of small to medium-sized, 20–
35 mm in diameter, putative nociceptive DRG neurons of adult rats.
We also examined the effect of BAB on the depolarization-evoked
firing of the DRG neurons
We hypothesized that Nav1.6 channels are less sensitive to BAB
than Nav1.7 and Nav1.8 channels, which would account for the
greater selectivity of BAB for sensory versus motor pathways, and
that BAB might produce a use-dependent block that would make
hyperactive neurons more sensitive to its suppressant action.
An understanding of the action of BAB on Na þ channels might
provide new insights into the pathophysiological mechanisms of
afferent sensitization and lead to new approaches for the treatment of neuropathic pain. The long-term and selective anesthesia
induced by BAB may be due to a faster entry in the slowinactivated state of Na þ channels and a better selectivity to Na þ
channels expressed in sensory neurons (Nav1.7 and Nav1.8) than

motor axons (Nav1.6).

2. Materials and methods
2.1. Cell culture and cell lines
The stable cell lines were grown in Dulbecco’s minimal essential medium (DMEM, Gibco BRL Life Technologies) supplemented
with fetal bovine serum (FBS, 10%), L-glutamine (2 mM), penicillin
(100 U/ml), streptomycin (10 mg/ml), and 75 mg/ml hygromycin
(Gibco BRL Life Technologies). The cells were incubated at 37 1C in
a 5% CO2 humidified atmosphere.
To study the effect of BAB on the various Na þ channels, we used
three different cell lines that stably express Nav1.6, Nav1.7, or Nav1.8.
Briefly, to generate the cell lines, HEK293 cells were transfected with
the pIRESneo3/Nav vector using calcium phosphate method. One day
after the transfection, the cells were divided at various dilutions. On
day 2, 800 mg/ml of neomycin was added. Three weeks after the
transfection, individual clones were tested using the patch-clamp
technique to ensure that they expressed the selected Na þ channels.
Clones that generated currents 4500 pA were selected for study,
and the neomycin concentration was lowered to 400 mg/ml. Two
days before the recordings, the cells were transfected with pIRES/

CD8/β1 to include the regulatory subunit. Cells that bound to CD8
antibody-coated beads (Dynabeads M-450 CD8-a) were considered
to express the β1 subunit and were selected for recording.
2.2. DRG neuron culturing and dissection
Experiments were performed on male Sprague-Dawley rats
purchased from Charles River Laboratories (Wilmington, MA).
Animals were housed under conditions 12:12 h light–dark cycle
and constant room temperature and humidity. Food and water
provided ad libitum. All experiments were performed according to
the guidelines of the Canadian Council on Animal Care and were
approved by the Animal Care Committee of Laval University.
Neurons were isolated from the L4-L5 DRG of adult male rats.
Briefly, freshly removed ganglia were de-sheathed and enzymatically digested at 37 1C for 20 min in DMEM containing 2 mg/ml
of type 4 collagenase (Worthington Biochemical Corp.). Trypsin
(2.5 mg/ml, Sigma) was then added and the neurons were incubated for an additional 15 min. The ganglia were then dissociated
mechanically by trituration using fire-polished Pasteur pipettes.
The cell suspension was centrifuged for 5 min at 200 g at room

159

temperature. The cells were re-suspended in DMEM containing
4 mg/ml of type 2 S trypsin inhibitor (Sigma), layered on 7.5% BSA
in Dulbecco’s phosphate buffered saline (DPBS), and centrifuged at
200 g for 5 min. After removing the supernatant, the pellet
containing the neurons was re-suspended in DMEM containing
10% heat-inactivated horse serum and 5% FBS. The neurons were
plated on poly-D-lysine-coated dishes and were kept in a 95% air–
5% CO2 incubator at 37 1C until the patch-clamp recordings were
performed 1–2 days later.
2.3. Whole-cell patch-clamp recording
Whole-cell Na þ currents in HEK293 cells were recorded using
an Axopatch 200B with the whole-cell configuration of the patchclamp technique (Molecular Devices). pClamp v9.0 or later
was used for the pulse stimulations and recordings (Molecular
Devices). Currents were filtered at 5 kHz, digitized at 100 kHz
using a Digidata 1200 series AD converter (Molecular devices), and
stored on a personal computer for later offline analysis. Series
resistance was compensated by 70–80%. When needed, linear leak
current artifacts were removed using on-line leak subtraction.
Fire-polished low-resistance electrodes (1 MΩ) were pulled from
8161 glass (Corning) and were coated with Sylgard (Dow-Corning)

to minimize pipette capacitance.
GigaOhm-seal recordings of Na þ currents were obtained in
DRG neurons using the whole-cell patch-clamp technique. Immediately before the recordings, the serum-containing medium was
replaced with current-clamp recording solution. APs were generated by 5 ms, 50–300 pA rectangular current pulse injections
followed by a 100 ms interpulse at the holding potential and then
a 600 ms pulse. In general, the sequence consisted of at least two
control recordings of evoked APs followed by pharmacological
studies where increasing concentrations of BAB were sequentially
applied prior to recording the APs. In some experiments, the time
courses of the effects of TTX and BAB were monitored by repeating
the above sequence at a stimulus intensity just above that required
to evoke an AP.
2.4. Solutions and reagents
For the voltage-clamp recordings from HEK293 cells, the bath
solution for the Nav1.8 and Nav1.6 current recordings contained
150 mM NaCl, 2 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, 10 mM
glucose, and 10 mM HEPES. The pH was adjusted to 7.4 with 1 M
NaOH. To reduce the voltage-clamp error due to the large current
evoked by Nav1.7, the extracellular solution was supplemented
with 20 mM NaCl and 130 mM choline chloride to reduce the

maximum current and increase the clamp speed. For the DRG
neuron recordings, the bath solution contained 35 mM NaCl,
105 mM choline chloride, 3 mM KCl, 1 mM CaCl2, 1.0 mM MgCl2,
10 mM glucose, 10 mM HEPES, and 100 nM CdCl2.
For the HEK293 cells, the intracellular pipette solution for
Nav1.6 and Nav1.7 contained 35 mM NaCl, 105 mM CsF, 10 mM
EGTA, and 10 mM HEPES. The pH was adjusted to pH 7.4 with 1 M
CsOH. Since Nav1.8 currents are small, the driving force was
increased by reducing the intracellular Na þ concentration to
5 mM (30 mM NaCl was replaced with 30 mM CsF). For the DRG
neurons, the intracellular pipette solution contained 10 mM NaCl,
140 mM CsF, 1 mM EGTA, and 10 mM HEPES. The pH was adjusted
to pH 7.3 with 1 M CsOH.
For the current-clamp recordings, the extracellular solution
contained 154 mM NaCl, 5.6 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2,
10 mM glucose, and 8.0 mM HEPES. The pH was adjusted to
7.4 with 1 M NaOH. The pipette (intracellular) solution contained
122 mM KCl, 10 mM NaCl, 1.0 mM MgCl2, 1.0 mM EGTA, and 10 mM
HEPES. The pH was adjusted to 7.3 with 1 M KOH.

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BAB was applied by superfusion using a ValveLink8.2s perfusion system (Automate Scientific) through a 250 mM needle. Fresh
stock solutions of BAB dissolved in EtOH was prepared weekly. The
stock solutions contained 0.1% EtOH and the desired concentration
of BAB. Due to the very low solubility of BAB in water, no
concentrations over 600 mM BAB were tested. All chemicals were
purchased from Sigma Aldrich.
2.5. Data analysis
The data were analyzed using a combination of pCLAMP software v10.0 (Molecular Devices), Microsoft Excel, and SigmaPlot
11.0 (Systat Software, Inc.). All P-values are two-tailed, and
P o0.05 was considered statistically significant. Statistical values
are expressed as means 7SEM. Statistical testing was carried out
using a stepwise procedure depending upon the number of groups
being compared. When only two means were compared, a twotailed t-test with unequal variances was used. When more than
two means were involved, a one-way analysis of variance was first
carried out to obtain a global test of the null hypothesis. If the
global P-value for the test of the null hypothesis was o0.05, we
performed post-hoc comparisons between the different groups
using the Holm-Sidak test.
The conductance was calculated using the following equation:
(GNa ¼INa/(Vm - Vrev), where GNa is the conductance, INa is the peak
current for the test potential Vm, and Vrev is the reversal potential
estimated from the current–voltage curve. Conductance values
were then fitted to a Boltzmann equation: I/Imax ¼1/(1 þ exp
((VþV1/2)/k)), where Imax is the maximal evoked current, V1/2 is
the voltage at which half of the channels are in the open state, and
k is the slope factor. Steady-state inactivation values were fitted
using a similar Boltzmann equation. Slow inactivation was fitted
with the sum of two exponential curves, with a fast time constant
(τf) and a slow time constant (τs).

3. Results
3.1. Effect of BAB on APs in isolated rat DRG neurons
APs were recorded from cultured lumbar DRG neurons ranging
from 20 to 35 mm in diameter (average of long and short axes). TTX
applied at the end of the experiments had little or no effect on
firing at a holding potential of 60 mV (data not shown) (Yamane
et al., 2007). When the resting potential was held at 60 mV, the
DRG neurons fired with a mean numbers of APs of 13.0 70.9 in
response to a long 600 ms, 0.2 pA depolarizing pulse (Fig. 1;
n ¼12). The superfusion of 1 or 10 μM BAB did not decrease the
AP (Fig. 2A). The superfusion of 100 mM BAB decreased the AP
generated by a 0.2 pA pulse from 13.0 70.9 to 9.0 71.7. The first
overshoot (OS) (Fig. 2C) was not significantly different. The superfusion of 10 mM BAB significantly reduced the maximum rate
of rise (Fig. 2D, P o0.01). The OS decreased quickly in a dosedependent manner in the presence of BAB (Fig. 2E). The superfusion of 1 mM BAB decreased the OS of the last evoked AP by
12.5 mV from 33.97 6.1 mV to 21.4 75.4 mV. The superfusion of
100 mM BAB led to a greater decrease in the OS (28.1 mV). 100 mM
BAB increase the duration of the AP from 1.43 ms to 1.52 ms
(Fig. 2F).

Fig. 1. Effect of BAB on firing. Representative AP traces of firing triggered by a
600 ms depolarizing pulse (control) (A), and in the presence of 1 mM BAB (B), 10 mM
BAB (C), and 100 mM BAB (D). (E) Shows the current-clamp protocol used to
generate single APs and the long spike trains in A to D.

and Nav1.8 were tested at 20 mV, 40 mV, and þ10 mV, respectively. Representative current traces from control experiments,
400 mM BAB superfusion and washout are shown in Fig. 3A–C.
The inset shows the holding potential and test pulse in each
experiment. Nav1.6 and Nav1.7 heterologously expressed in
HEK293 cells displayed a similar block in the presence of 100 mM
BAB (15 72% for Nav1.6, 18 75% for Nav1.7), while Nav1.8 displayed
the highest degree of inhibition (30 74% block), which was
significantly different from the inhibition of Nav1.6 (P o0.05) and
Nav1.7 (P o0.01). In the presence of 600 mM BAB, Nav1.8 also
displayed a significantly greater inhibition (80 74% block) than
that of Nav1.6 (42 73%) and Nav1.7 (497 3%) (P o0.01).
3.3. Effect of BAB on the activation of heterologously expressed Na þ
channels

3.2. Tonic block by BAB of Na þ channels expressed in HEK293 cells
The tonic effect of BAB on heterologously expressed Na þ
channels in HEK293 cells was tested using 40 ms depolarizing
pulses at a voltage that evoked the maximum current. The voltage
was different for each Na þ channel subtype (Fig. 3). Nav1.6, Nav1.7,

Fig. 4A–C shows the effect of BAB on the voltage-dependence
of activation of Nav1.6, Nav1.7, and Nav1.8, respectively. Values
are plotted as relative membrane conductances as explained in
Materials and methods. The different biophysical properties of the
Na þ channel subtypes meant that the voltage protocols had to be

O. Thériault et al. / European Journal of Pharmacology 727 (2014) 158–166

161

Fig. 3. Concentration-dependent suppression of Nav1.6, Nav1.7, and Nav1.8 currents
by different concentrations of BAB. Whole-cell Na þ currents in HEK293 cells were
evoked by a 40 ms depolarizing pulse to -20 mV for Nav1.6, -40 mV for Nav1.7, and
þ10 mV for Nav1.8 (holding potential of -140 mV). (A–C) Representative tracecurrents of Nav1.6, Nav1.7, and Nav1.8, respectively, in control conditions (gray),
400 mM of BAB (black) and washout (dotted line). (D) Representation of the relative
inhibition by BAB of the different Na þ channels. The inset of (D) shows concentration–response curves for Nav1.6 (filled circle), Nav1.7 (open circle), and Nav1.8
(filled triangle). (*P o0.05; **Po 0.01; n¼ 4–10).
Fig. 2. Effect of BAB on AP parameters. In a separate series of experiments, a
200 pA current injection (as shown in Fig. 1E) was used to generate single APs and
long 600 ms spike trains, and the following parameters were measured: AP no./
600 ms (A); voltage-threshold for firing (B); OS (C); maximum rate of rise, dV/dtmax
(D); last generated OS/first OS (E); and duration of AP at 50%, AP50 (F). 1, 10, and
100 μM BAB applied in that sequence. (np 40.01, nnp 40.001, n¼12).

are shown in the insets. BAB did not alter the voltage dependence
of the Na þ channels (see Table 1).

adjusted for each subtype. Briefly, short 50 ms depolarizing pulse
was applied in increments starting from a holding potential at
140 mV. Pulses ranging from
80 mV to þ90 mV in 5 mV
increments were used for Nav1.6 (Fig. 4A) and Nav1.8 (Fig. 4C)
and from 90 mV to þ15 mV for Nav1.7 (Fig. 4B). The protocols

The voltage-dependences of Nav1.6, Nav1.7, and Nav1.8 was
determined using the protocols shown in the insets in Fig. 4D–F.
The values were then fitted with a Boltzmann equation. For Nav1.6,
a conditioning pulse was applied from
150 mV to
5 mV
in 5 mV increments followed by a test pulse to
20 mV. BAB

3.4. Effect of BAB on fast inactivation

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O. Thériault et al. / European Journal of Pharmacology 727 (2014) 158–166

Fig. 4. Effect of BAB on the steady-state activation and inactivation of Nav1.6, Nav1.7, and Nav1.8. (A–C) Steady-state activation of Nav1.6, Nav1.7, and Nav1.8, respectively, in the presence
of 100 mM BAB (open triangles) or in control conditions (filled circles). The stimulus protocols are shown in the figure insets. Conductance was derived from the maximum amplitude
for each voltage obtained from the IV curves. (D–F) Steady-state inactivation of Nav1.6, Nav1.7, and Nav1.8, respectively. Steady-state inactivation was determined using 500 ms
conditioning pulses followed by a standard test pulse. The test current was normalized and plotted against the conditioning voltage. The voltages are indicated in the protocol shown in
the inset of each panel. Control condition (filled circles) and 100 mM BAB (open triangles). Values and significance are listed in Table 1. See Material and methods for details.

(100 mM) caused a 17.8 mV hyperpolarizing shift in the V1/2
from
71.575.1 mV in the control condition to
89.372.6 mV
after the superfusion of BAB (Fig. 4D). The slope factor was also
significantly shifted from 5.170.2 to 6.170.4. For Nav1.7, conditioning pulses were applied from 150 mV to 35 mV, and currents
were determined with a test pulse to 40 mV (Fig. 4E). Fig. 4F shows
the inactivation curve of Nav1.8 determined with a conditioning
pulse from 140 mV to þ5 mV and a test pulse to 0 mV. Nav1.7 and
Nav1.8 also exhibited significant hyperpolarizing shifts of 17.1 mV
(Po0.01) and 9.6 mV (Po0.05), respectively. The effects of BAB on
the parameters of inactivation are summarized in Table 1.

consisted of a conditioning pulse of variable duration (1 ms to 10 s)
followed by a 10 ms pulse to allow for recovery from fast inactivation
and then a 40 ms test pulse. The conditioning pulse and the test pulse
were to 10 mV for Nav1.6, 20 mV for Nav1.7, and þ 15 mV for
Nav1.8. BAB did not affect the time constants of Nav1.6 slow inactivation (Fig. 5A). The slow inactivation curve of Nav1.7 was much steeper
in the presence of 100 mM BAB, which was mainly a result of a marked
(50%) reduction in the slow time constant (τs was 742871006 ms in
the control and 36587264 ms after the superfusion of 100 mM BAB)
(Fig. 5B). A similar effect was observed for Nav1.8 (Fig. 5C), which was
also due to an acceleration of the slow time constant, with little or no
change in the rapid time constant (Table 1).

3.5. Effect of BAB on slow inactivation
3.6. Frequency-dependent block
We previously showed that the local anesthetic lidocaine
differentially modulates the slow inactivation of Nav1.7 and
Nav1.8 (Chevrier et al., 2004). In the present study, we tested the
effect of BAB on slow inactivation using a similar protocol, which

Stimulation frequencies up to 20 Hz were used to test the
frequency-dependent block of Na þ channels by BAB (100 mM).
Fifty stimulus pulses were applied at the voltage that elicited the

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163

Table 1
Effects of BAB on fast activation, inactivation and slow inactivation parameters.
Nav1.6 (HEK293)
Control
Activation
V1/2 (mV)
kv
n

8

Inactivation
V1/2 (mV)
kv
n

71.5 7 5.1
5.17 0.2
8

Nav1.7 (HEK293)
100 mM BAB

35.871.4
6.17 0.3

Slow-inactivation
τf (ms)
2069 7 683
τs (s)
18.8 73.5
n
4

38.4 7 1.8(NS)
6.3 7 0.4(NS)

Control

Nav1.8 (HEK293)
100 mM BAB

Control

7

48.17 1.5
5.5 70.3
10

48.57 3.2(NS)
6.0 70.3(NS)
4

89.3 7 2.6b
6.1 70.4 b
6

92.8 72.0
7.4 7 0.8
10

109.9 7 2.4
7.0 7 0.4(NS)
4

871 7285(NS)
19.17 5.2(NS)
5

70.3726.6
7.4 7 1.0
4

76.17 5.5(NS)
3.6 7 0.26b
6

b

(DRG)
100 mM BAB

Control

16.2 7 3.1(NS)
13.0 7 0.8(NS)

100 mM BAB

21.6 71.3
12.0 7 0.4
31

29.0 71.1
5.0 7 0.5

24.17 5.1(NS)
6.4 70.8(NS)

7

6

5

58.5 7 1.6
7.9 7 0.7
10

68.17 3.8a
7.8 7 0.7(NS)
7

38.8 70.9
4.3 7 0.2
7

54.7 72.5b
4.5 7 0.2(NS)
9

248 7 85
43.6 76.1
5

173 715(NS)
29.2 73.4a
7

–
–
–

–
–
–

τf ¼ fast inactivation time constant; τs ¼ slow inactivation time constant; n¼ number of experiments; NS: not significant.
a
b

P o0.05.
Po 0.01.

maximum current for each channel subtype ( 20 mV for Nav1.6,
40 mV for Nav1.7, and þ 10 mV for Nav1.8). Currents were
normalized to the first pulse in the sequence. Nav1.6 currents
exhibited a slight frequency-dependent block in the presence
of BAB. At 20 Hz there was a further reduction in the normalized
current (Fig. 6A, 7.5%, P o0.05). Nav1.7 displayed the highest
sensitivity to the frequency-dependent block (Fig. 6B, a significant
7% reduction at 10 Hz (P o0.01) and a 20% reduction at 20 Hz
(P o0.01)). The frequency-dependent block of Nav1.8 was similar
to that of Nav1.6 (Fig. 6C, 7.4% at 20 Hz).
3.7. Effect of BAB on the TTXr Naþ channels of rat DRG neurons
The tonic block of TTXr Na þ currents was measured using a
50 ms test pulse to 0 mV that was repeated every 10 s until
the current reached a steady-state which occur between 3 and
5 min. BAB was applied sequentially at concentrations of 1, 10, and
100 μM, and the steady-state inhibition at each concentration was
compared to the control current before applying BAB (Fig. 7A,
n ¼10). The inhibition of the TTXr Na þ currents of DRG neurons
was greater (48 76%, Fig. 7A) than the inhibition (31 74%, Fig. 3D)
of Nav1.8 currents in HEK293 cells.
As observed with HEK293 cells expressing Nav1.8, 100 mM
BAB had no effect on the voltage-dependence of activation of TTXr
Na þ currents (Fig. 7B). However, 100 mM BAB provoked a 16 mV
(P o0.001) hyperpolarized shift of the voltage-dependence of
inactivation of the TTXr Na þ current of DRG neurons compared
to 10 mV in transfected cells (Table 1).
The frequency-dependent block of the TTXr Na þ current of
DRG neurons was studied by adjusting the frequency of stimulation from 2 to 20 Hz in the absence or presence of 100 mM BAB
(Fig. 7C). At 10 Hz, there was a 5% increase in the frequencydependent inhibition of the TTXr Na þ current of DRG neurons
(p 40.01), while at 20 Hz, the inhibition increased to 9%.

4. Discussion
The experiments described in the present study were designed
to shed light on the mechanisms underlying the prominent
analgesic effect of epidural BAB that occurs with relatively few
adverse effects. We observed substantial differences in the BAB
sensitivity of the three types of currents generated by Na þ

channels expressed in HEK293 cells. TTXr Na þ currents (Nav1.8)
in these neurons were also sensitive to BAB at similar concentrations. Currents generated by TTXr Nav1.8 channels expressed in
HEK293 cells were also more sensitive to BAB than currents
generated by TTXs Nav1.6 and Nav1.7 channels, although the
heterologously expressed channels were less sensitive to BAB than
native channels. An analysis of the frequency and time dependent
inactivation of currents also revealed differences in the effect of
BAB on the different channels. These results suggested that the
clinical usefulness of epidural BAB in treating pain may be related
to the targeting of specific subtypes of Na þ channels in sensitized,
small diameter, nociceptive afferent neurons.
The clinical efficacy occurs with a series of four epidural
injection of 5% BAB in suspension (Shulman et al., 1998). A study
on the diffusion of few local anesthetics through the human duraarachnoid supports the hypothesis that the selective action of BAB
suspension can be attributed to the spatial confinement into the
epidural space (Grouls et al., 2000). The prolonged analgesia
produced by BAB can in large part be attributed to the physiochemical properties of the drug (water solubility, partition coefficient) that enable its formulation as a hydrophobic suspension.
After injection into the epidural space BAB slowly leaches out of
suspension onto the adjacent nerve roots thereby producing a
selective inhibition of sensory nerve fibers.
In DRG neurons, concentrations of BAB as low as 1 mM elicited
prominent changes in the OS of the last AP evoked by depolarizing
current pulses and in the tonic block. The effect in the OS of
the last AP evoked is most likely because more Na þ channels enter
the slow inactivated state in the presence of BAB. At least
part of this effect of BAB on nociceptive neuron excitability may
be due to its effect on TTXr Na þ channels. Indeed, BAB produced a
concentration-dependent steady-state inhibition and a frequencydependent inhibition of TTXr Na þ currents and a hyperpolarizing
shift in the inactivation curve of the TTXr Na þ currents in
these neurons. These effects occurred with no change in the
activation curve. Furthermore, we showed that the effects of BAB
are completely reversible within a minute after the washout on
sodium channels expressed in HEK293 cells.
However, it has been reported that BAB affects multiple ion
channels, including a block of potassium channels (Beekwilder
et al., 2003;Winkelman et al., 2005), which suggests that it may
depolarize the resting membrane potential and lead to greater Na þ
channel inactivation (slow and fast). Calcium channels (Beekwilder

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O. Thériault et al. / European Journal of Pharmacology 727 (2014) 158–166

Fig. 5. Impact of BAB on the slow inactivation of Nav1.6, Nav1.7, and Nav1.8. (A–C)
The slow activation of Nav1.6 (A), Nav1.7 (B), and Nav1.8 (C) was studied in control
condition (filled circles) and in the presence of 100 mM BAB (open triangles). The
entry into the slow inactivation state was measured using a double-pulse protocol
consisting of a conditioning first test pulse of variable duration (1 ms to 10 s), a
10 ms interpulse to 140 mV, and a second test pulse. The voltages used in the first
and second test pulses were 10 mV for Nav1.6, 20 mV for Nav1.7, and þ15 mV
for Nav1.8. The currents were normalized and were plotted against the duration of
the conditioning pulse. The values were fitted with the sum of two exponentials in
all cases. See Table 1 for the time constant values.

et al., 2005) and TRP channels (Bang et al., 2012) have also been
reported to be blocked by BAB. It is thus possible that the effect on AP
parameters results from a combination of effects on Na þ and other
ion channels.
To compare the effects of BAB on TTXr and TTXs Naþ channels,
we expressed the channels in HEK293 cells. In the absence of BAB,
we observed significant differences in the biophysical properties of
native TTXr Na þ currents (presumed to reflect primarily Nav1.8)
recorded in DRG neurons and the Nav1.8 currents recorded in
HEK293 cells. The native TTXr Na þ currents exhibited a significant
8 mV hyperpolarized shift in activation parameters and an even
greater 20 mV depolarizing shift in inactivation parameters. We
also observed a significant difference in the frequency dependent
block above 10 Hz between TTXr Na þ currents of DRG neurons

Fig. 6. Use-dependent inhibition of Nav1.6, Nav1.7, and Nav1.8 by BAB. Currents
were evoked by test pulses at different frequencies. The black columns are the
control condition and the gray columns are in presence of 100 mM BAB. Test pulses
were 20 mV for Nav1.6 (A), 40 mV for Nav1.7 (B), and þ 10 mV for Nav1.8 (C).
See the inset for the protocols. (nPo 0.05; nnP o 0.01; n ¼5–7).

and Nav1.8 transiently expressed in HEK293 cells. The reason for
the differences between heterologously expressed Na þ channels
and DRG Na þ channels is uncertain, but may be due to other
regulatory processes in native tissue but absent in HEK293 cells.
However, BAB had a similar effect on Nav1.8 channels in DRG
neurons and Nav1.8 channels heterologously expressed in HEK293
cells despite the differences in basal biophysical properties. The
shift in inactivation parameters and the frequency-dependent
inhibition caused by BAB was similar for native TTXr currents
and Nav1.8 currents in HEK293 cells.
A difference in the affinity of BAB for the different Na þ channel
subtypes does not entirely explain why BAB causes selective
analgesia without reducing motor function or touch perception.
We only observed a small tonic block of the total Na þ current in
transfected cells with 100 mM BAB (18% Nav1.7, 31% Nav1.8, and 15%
Nav1.6). Since the affinity is low and the inhibition is probably
partial, it was not possible to extrapolate these data to an IC50 for
BAB or explain the clinical efficacy of BAB based on differences in
the tonic blocks of Nav1.8, Nav1.6, and Nav1.7 channels stably

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165

The role of slow inactivation in nociceptive fibers is relatively
well known and appears to be important in neuronal excitability.
A mutation in Nav1.7 that reduces the kinetics of slow inactivation
has been reported to exacerbate pain in patients with small fiber
neuropathy (Han et al., 2012). Furthermore, the entry of Nav1.8
into slow inactivation reduces firing in small diameter DRG
neurons (Blair and Bean, 2003). It has also been reported that
molecules that stabilize Na þ channels in the slow inactivated state
attenuate neuropathic pain (Hildebrand et al., 2011). It is thus
likely that the increase in the onset of slow inactivation of Nav1.7
and Nav1.8 in the presence of BAB contributes to the anesthesia
induced by this drug.
Nav1.6 is thought to be a major component of the motor axon
AP. It is also preferentially expressed in sensory A-fibers and is
localized at the nodes of Ranvier, dendrites, and synapses
(Fukuoka et al., 2008;Caldwell et al., 2000). Nav1.6 has a significantly lower affinity than Nav1.8 for BAB and the onset of slow
inactivation of Nav1.6 is not affected at all by BAB, both of which
might, in part, explain its selectivity.
In summary, we propose that the mechanism by which BAB
induces long-term anesthesia may include an effect on the slow
inactivation of Na þ channels. The selectivity of the anesthetic may,
in part, be due to more pronounced effects on channels expressed
in small-medium diameter nociceptive sensory neurons (Nav1.7
and Nav1.8) than on channels expressed in large diameter nonnociceptive sensory neurons and motor axons (Nav1.6).
Acknowledgments
This work was supported by grants from the Canadian Institutes of Health Research (CIHR, MT-13181); the Heart and Stroke
Foundation of Quebec (HSFQ).
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Fig. 7. Effect of BAB on the TTXr Na þ channels of DRG neurons. (A) Tonic block of
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